The present invention relates to a novel D-hydantoinase from Ochrobactrum anthropi that enantio-selectively hydrolyzes D-hydantoins to their corresponding D-N-carbamoyl-xcex1-amino acid; the nucleic acid that encodes for the enzyme; an expression vector including the nucleic acid; and a host cell capable of expressing the enzyme.
Hydantoinase is an enzyme that catalyzes the conversion of 5-monosubstituted hydantoins to the corresponding N-carbamoyl-xcex1-amino acids. The optically pure N-carbamoyl-xcex1-amino acids can then be hydrolyzed by chemical or enzymatic means to amino acids. This important entantioselective property makes them valuable for the production of optically pure D- or L-amino acids, which are useful intermediates for the preparation of semisynthetic penicillins and cephalosporins. The use of hydantoinase to produce optically pure N-carbamoyl amino acids is known in the art [Syldatk, C., Muller, R., Siemann, M., Krohn, K., and Wagner, F. (1992). In Biocatalytic production of amino acids and derivatives. (D. Rozell and F. Wagner, Ed.) p.75-128 Hanser Publishers, New York]. Hydantoinase enzymes have been isolated from a variety of sources including Klebsiella, Corynebacterium, Agrobacterium, Pseudomonas, Bacillus, and Streptomyces. European Patent Application EP/0739978 A2 describes a hydantoinase produced from Agrobacterium tumefaciens exhibiting improved activity and stability in alkaline medium at high temperatures. U.S. Pat. No. 5,516,660 discloses novel specimens of the Arthrobacter species which produce hydantoinases that are capable of producing L-xcex1-amino acids from D-, L- and/or D,L-5-monosubstituted hydantoins. U.S. Pat. No. 5,714,355 describes a mutant specimen of the Arthrobacter species which has up to 2.7 fold greater enzymatic activity than the parent organism. PCT Publication WO 00/58449 describes modified hydantoinases that exhibit improved enzymatic properties relative to previously isolated hydantoinases. There still remains a need to isolate new enzymes that exhibit improved enantioselectivity as well as catalytic activity.
The present invention is directed to isolated and purified D-hydantionase from Ochrobactrum anthropi preferably having the sequence of SEQ ID NO: 2 or a protein having at least 80% identity to SEQ ID NO: 2.
The present invention is also directed to nucleic acids coding for the enzyme, preferably the genomic DNA of SEQ ID NO: 1 or cDNA derived therefrom.
The present invention is also directed to a recombinant host cell comprising nucleic acid coding for the enzyme of the invention.
The present invention is also directed to an expression vector comprising nucleic acid coding for the enzyme of the invention.
Further, the present invention is directed to a method for producing the enzyme of the invention comprising culturing a suitable cell containing nucleic acid coding for the enzyme of the invention in a suitable medium under conditions suitable for expression of the enzyme.
Finally, the present invention is directed to a process for converting 5-monosubstituted hydantoins to the corresponding N-carbamoyl-xcex1-amino acids using the hydantoinase of the invention to result in a product of high chiral purity.